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Abstract

Background

MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers.

Results

The sensitivity of the methylated-MSP was one in 103. Different MSP statuses, including complete methylation, partial methylation, and
complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All
five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM
at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression
(p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated
for MIR129-2, led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular
proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation.

Conclusions

MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid
malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In
NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis.

Keywords:

Background

DNA methylation, which adds a methyl group to the number 5 carbon of a cytosine ring
of a CpG dinucleotide, is catalyzed by DNA methyltransferase [1,2]. Cancers are characterized by a global DNA hypomethylation and locus-specific hypermethyla-tion
of tumor suppressor gene (TSG). Based on a pathway-specific approach, multiple TSGs
in pathways including cell cycle regulation, Janus kinase/signal transducer and activator
of transcription (JAK/STAT) signaling, wingless-type MMTV intergration site family
(WNT) signaling, and death-associated protein (DAP) kinase-associated intrinsic tumor
suppression, have been shown to be inactivated by gene hypermethylation in leukemia,
lymphoma and multiple myeloma (MM) [1,3].

MicroRNAs are short sequences (22–25 nucleotides) of non-coding RNA molecules that
regulate a range of biological processes by inducing RNA degradation and/or translation
inhibition of targeted mRNAs [4]. Precise microRNA expression is commonly dysregulated in human diseases, including
cancers. In carcinogenesis, of these aberrantly expressed microRNAs in malignant cells,
those upregulated microRNAs which lead to targeting of tumor suppressor genes are
known as oncomiRs. On the other hand, those downregulated microRNAs which originally
may inactivate oncogenes are known as tumor suppressive microRNAs [5,6]. Recently, DNA methylation has emerged as an important mechanism in the regulation
of microRNA expression, in particular, hypermethylation of microRNA gene promoters
may lead to inactivation of tumor suppressive microRNAs in cancers [7].

In human, MIR129 is transcribed from MIR129-1 and MIR129-2 located on chromosome 7q32 and 11p11 respectively. A CpG island is present in the
proximity of MIR129-2 but not MIR129-1 promoter. Moreover, loss of MIR129 expression by MIR129-2 methylation has been reported in gastric, endometrial, and colorectal cancers [8-10], leading to upregulation of oncogenes including cyclin-dependent kinase 6 (CDK6) and sex determining region Y-box 4 (SOX4) mRNAs, thereby illustrating the tumor suppressive effect of MIR129[9-12].

Figure 1.Methylation of MIR129-2. (A) Schematic diagram showing the distribution of CpG dinucleotides (solid vertical
lines) along precursor MIR129-2 and its upstream promoter region. Region of MSP amplification was indicated by the
text box marked as “MSP”. Sequence analysis of the MIR129-2 M-MSP product from bisulfite-treated methylated control DNA showed that the cytosine
[C] residues of CpG dinucleotides were methylated and remained unchanged, whereas
all the other C residues were unmethylated and were converted to thymidine [T], indicating
complete bisulfite conversion and specificity of MSP. (B) Sensitivity of the methylated-MSP for the MIR129-2. (C) M- & U-MSP of 15 healthy donor controls showing no MIR129-2 methylation. (D) All myeloma cell lines, except RPMI-8226, were partially methylated, while RPMI-8226
was completely unmethylated for MIR129-2. (E) All five lymphoma cell lines were completely methylated for MIR129-2.

There was no MIR129-2 methylation detected in any of the AML and CML patients (Figure 2A). In ALL, MIR129-2 methylation was detected in only 1 (5%) of 20 patients. In CLL, MIR129-2 methylation occurred in 28 (45.9%) patients (Figure 2A). MIR129-2 methylation was not correlated with mean or median hemoglobin level, lymphocyte and
platelet counts. Moreover, there was no correlation between MIR129-2 methylation and age, gender, Rai stage (≥stage 2), or high-risk karyotypic aberrations.
In 50 CLL patients with concomitant data on MIR34A, MIR124-1, MIR196B and MIR203 methylation, there was no association with MIR129-2 methylation with methylation of these microRNAs (data not shown). On the other hand,
the median survivals were significantly inferior in patients with MIR129-2 methylation than those without (49 versus 111 months, p=0.004; Figure 2B).

In MM, MIR129-2 was methylated in 47 patients (49.5%) at diagnosis and 12 patients (41.4%) at relapse
(p=0.590). Methylation of MIR129-2 was more frequent in IgD immunoglobulin isotype, occurring in 3 (100%) of IgD, 34
(59.5%) of IgG, 8 (34.8%) of IgA, and 2 (18.2%) of light chain but none of non-secretary
MM (p=0.013). However, there was no association of MIR129-2 methylation with gender, ISS, median OS, or methylation of MIR34A, MIR124-1, MIR196B or MIR203. Interestingly, MIR129-2 methylation was only detected in 11 (27.5%) patients with MGUS. Therefore, MIR129-2 methylation was more frequent in MM at diagnosis than patients with MGUS (p=0.023).

5-azadC treatment and MIR129 overexpression in lymphoma cell lines

To investigate if the MIR129-2 methylation might lead to low MIR129 expression, JEKO-1 with homozygously methylated MIR129-2 was treated with different concentrations of 5-azadC for 3 days and tested for MSP
and stem-loop RT-qPCR. On treatment with 5-azadC, MIR129-2 was demethylated (Figure 4A; Additional file 1: Figure S4), with a corresponding increase in MIR129 expression (Figure 4B) and downregulation of SOX4 mRNA (Figure 4C), which has been shown to be a direct target of MIR129. To validate the tumor suppressive effect of MIR129, MIR129 was overexpressed in JEKO-1 cells (Figure 5A). Upregulation of MIR129 led to SOX4 downregulation (p=0.036, Figure 5Aii). Furthermore, MIR129 expression resulted in reduction of cellular proliferation as measured by MTT assay
(p=0.018, Figure 5Aiii) and an increase of dead cells as measured by trypan blue exclusion assay (p=0.017,
Figure 5Aiv). The tumor suppressive effect of MIR129 in the inhibition of cell proliferation and enhancement of cell death was further
demonstrated in GRANTA-519 cells, which is also completely methylated for MIR129-2 (Figure 5B).

Discussion

In this study, we demonstrated that MIR129-2 was hypermethylated in NHL and MM cell lines but not in normal blood or mononuclear
cells, illustrating a methylation pattern similar to other epigenetically silenced
tumor suppressor microRNAs, such as MIR34A, MIR34B/C, MIR124, and MIR203, in hematological cancers [13-17]. This is in contrast to some methylated microRNAs, such as MIR127 and MIR373, which show a tissue-specific methylation pattern, with methylation occurring in
both tumor cells and their normal counterparts [18]. Moreover, methylation leading to reversible gene silencing was illustrated here
with re-expression of MIR129 upon hypomethylation of MIR129-2. Furthermore, overexpression of MIR129 led to decreased cell proliferation with increased cell death. These results were
consistent with a tumor suppressor role of MIR129 in lymphoma cells, similar to its effects on other epithelial cancers [9-11,19]. In particular, SOX4, a known target of MIR129, facilitates differentiation of lymphocytes, and has been shown upregulated in various
human cancers [20]. Indeed, herein, downregulation of SOX4 was shown associated with upregulation of MIR129 upon either hypomethylating treatment or overexpression in GRANTA-519 and JEKO-1
lymphoma cell lines. Taken together, the findings indicate that hypermethylation of
MIR129-2 led to reversible inactivation of tumor suppressive MIR129 in hematological cancers. Lastly, in cell lines which showed complete methylation
of MIR129-2, there is no deletion of the MIR129-2 locus, i.e. chromosome 11p11 [21], and hence complete methylation in these cells suggests biallelic MIR129-2 methylation.

Secondly, we found that MIR129-2 methylation was frequent and appeared to be associated with poor survival in CLL
patients, which warrants future prospective studies with larger number of patients.
In CLL, apart from ZAP-70 gene hypermethylation being a favourable prognostic marker, there is little information
on the role of DNA methylation in the pathogenesis and clinical outcome of the disease
[22-26]. Furthermore, understanding of the prognostic value of microRNA and microRNA methylation
in CLL remains preliminary [14-16,27-29]. Hence, our observation of MIR129-2 methylation adversely impacting on survival in CLL is a novel finding. In order to
establish the prognostic significance of MIR129-2 methylation in CLL, a multivariate analysis together with Rai stage, lymphocyte counts
and high-risk karyotype is required. However, the small number of patients in this
cohort precluded a multivariate analysis.

In NHL, in contrast to MIR34A, MIR124-1, and MIR203, which were frequently methylated in NK- or B-cell lymphoma, MIR129-2 methylation was frequent but comparable among B-, T- or NK-cell lymphomas. However,
an interesting observation was that methylation of MIR129-2, which is localized to chromosome 11p11, was associated with methylation of MIR124-1 (localized to 8p23) and MIR203 (localized to 14q32). As MIR124-1 targets CDK6 mRNA and MIR203 targets ABL and CREB mRNAs, the strong association of methylation of these microRNAs suggested collaboration
of silencing of multiple microRNAs for oncogenesis [14,16]. Moreover, in lymphoma samples, in which both DNA and RNA were available, significantly
lower expression of MIR129 was demonstrated in primary lymphoma samples with MIR129-2 methylation than those without, further testifying the association of microRNA silencing
with microRNA hypermethylation.

In MM, MIR129-2 methylation was more frequent in MM patients at diagnosis or relapse/progression
than patients with MGUS, and hence might be an important event implicated in transformation
of MGUS to symptomatic MM. Despite that these samples were not CD138-sorted, the mean
and median of plasma cell percentage of these MGUS samples were 4.78 and 5 respectively,
and hence well within the limit of detection by the M-MSP. However, MSP performed
on CD138-sorted plasma cells would be ideal, and hence the current finding warrants
further studies using CD138-sorted samples. On the other hand, there was no impact
of MIR129-2 methylation on OS. However, this cohort of patients was heterogeneously-treated,
and hence the prognostic impact of MIR129-2 methylation remains to be verified in a cohort of uniformly-treated patients.

Conclusions

In summary, MIR129 is a putative tumor suppressive microRNA, and methylated in a tumor-specific manner,
leading to reversible microRNA silencing. MIR129-2 methylation was frequent in lymphoid but uncommon in myeloid neoplasms. In CLL, MIR129-2 methylation adversely impacted on survival. In NHL, MIR129-2 methylation was associated with methylation of other tumor suppressor microRNAs.
In MM, MIR129-2 methylation was probably associated with progression from MGUS to symptomatic MM.
Therefore, MIR129-2 methylation is important in the pathogenesis, disease progression and prognostication
in lymphoid neoplasms. The implication of MIR129-2 methylation with methylation of other tumor suppressive microRNAs in lymphomas warrants
further study.

Diagnosis of leukemia and lymphoma were made according to the French-American-British
Classification and WHO Classification of Tumors respectively [30-33].

In the CLL group, median overall survival (OS) was 81 months for the whole group,
and 102 months in those with limited, and 54 months in those with advanced Rai stage
(p=0.009). Median OS of CLL patient with low/standard-risk and high-risk karyotypes
were 111 months and 21 months (p<0.001).

In the NHL group, of 36 patients with data available at clinical presentation, 23
had nodal and 13 had extranodal involvement. Correlation between microRNA methylation
and expression was studied in 25 primary lymphoma samples (follicular lymphoma, N=12;
diffuse large B-cell lymphoma, N=13), in which both DNA and RNA were available.

The diagnosis of MGUS and MM was based on standard criteria [34]. Complete staging work-up included bone marrow examination, skeletal survey, serum
and urine protein electrophoresis, and serum immunoglobulin (IgG, IgA, and IgM) levels.
In this cohort, the median OS was 44 months, and projected 10-year OS was 19.9%. The
median OS were 83 months, 60 months and 23 months in those with ISS I, II and III
disease respectively (p<0.001). Definitions of relapse and disease progression followed
the criteria of European Group for Blood and Marrow Transplantation Registry [35]. Briefly, “relapse” from complete remission (CR) was defined as the reappearance
of the same paraprotein detected by serum/urine protein electrophoresis, appearance
of new bone lesion or extramedullary plasmacytoma, or unexplained hypercalcaemia.
The definition of “disease progression” from plateau phase/stable disease was the
same as the definition of relapse except that “a >25% increase in paraprotein level”
replaced “reappearance of the same paraprotein”. The study has been approved by Institutional
Review Board of Queen Mary Hospital, and written informed consent was obtained from
the patient for publication of this report and any accompanying data or images.

Hypomethylating treatment

JEKO-1 was homozygously methylated for MIR129-2. Cells were seeded in six-well plates at a density of 1x106 cells/ml and cultured with 0.5–1uM of 5-aza-2’-deoxycytidine (5-azadC) (Sigma–Aldrich)
for 3 days.

Quantitative real-time reverse transcription–PCR (RT-qPCR)

Short mature microRNA transcripts were quantified using stem-loop RT-qPCR which is
a sensitive, specific and widely-used method designed for microRNA studies [36]. For MIR129, RT was performed using Taqman® MicroRNA RT Kit and Taqman® MicroRNA Assay Kit (ABI,
Foster City, CA, USA), according to the manufacturer’s instructions. Total RNA was
reverse transcribed in 1 mmol/l dNTPs, 50 U MultiScribe™ Reverse Transcriptase, 1×
RT Buffer, 3·8 U RNase Inhibitor, and 1× stem-loop RT primer at following thermal
cycling condition: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. RT-qPCR of
MIR129 was performed using 1·33 μl of 1:15 diluted RT product in 1× Taqman® Universal PCR
Master Mix, and 1× Taqman® Assay at 95°C for 10 min, followed by 40 cycles of 95°C
for 15 s and 60°C for 1 min. SNORD48 was used as reference for data analysis with the 2-ΔΔCt method [37]. Conventional RT-qPCR was used for SOX4 transcript, RT was performed using QuantiTect Reverse Transcription Kit (Qiagen),
according to the manufacturer’s instructions. RT-qPCR was performed by iQ SYBR Green
Supermix (Bio-Rad), using GAPDH as endogenous control for data analysis with the 2-ΔΔCt method [37]. Primers for detecting SOX4 and GAPDH were summarized in Table 2.

MIR129 overexpression in JEKO-1 cells

Cells at log phase were transfected with 150nM of either negative control mimic or
MIR129 oligo mimic (Ambion) at a density of 106 cell/mL using X-tremeGENE siRNA transfection reagent (Roche), according to the manufacturer’s
instructions.

MTT assay

Cell proliferation was determined by colorimetric quantification of purple formazan
formed from the reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,
5-diphenyltetrazolium bromide) by proliferating cells. Briefly, cells were seeded
in a 96-well microtitre plate at 5 × 105 /well in 100 μl of medium. At the assay time point, each well was added 10 μl of
5 mg/ml MTT reagent (Sigma-Aldrich), followed by 6-hour incubation, after which 100 μl
of DMSO was added. The absorbance reading at 550 nm with reference to 650 nm was recorded.
Relative abundances of proliferative viable cells from three independent experiments
were calculated.

Trypan blue exclusion assay

Dead cells were visualized by trypan blue staining and five random microscopic fields
were counted for each sample. Dead cells (%) = (total number of dead cells per microscopic
field/ total number of cells per microscopic field) X 100. Percentages of dead cells
from three independent experiments were calculated.

Statistical analysis

Correlation between MIR129-2 methylation with continuous (mean age) and categorical variables (gender, histological
subtypes, lineage [B, T or NK/T] and nodal/extranodal presentation) were studied in
these 68 patients by Student’s t-test and Chi-square test (or Fisher Exact test) respectively. Overall survival (OS)
was measured from the date of diagnosis to the date of last follow‐up or death. Survival
was plotted by the Kaplan‐Meier method, and compared by the log‐rank test. Moreover,
in 25 primary B-cell NHL samples in which both DNA and RNA were available, the mean
expression of MIR129 in methylated and unmethylated lymphoma was compared by the Student’s t-test. Association
between MIR129-2 methylation and other previously studied tumor suppressive microRNA methylation,
including MIR34A, MIR124-1, MIR203 and MIR196B[14-16], in MM, NHL and CLL patients were studied by Χ2 test. The mean results from triplicate experiments after MIR129 transfection were compared by Student’s t-test. All p-values were 2-sided.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

CSC designed the study. KYW, RLHY conducted the experiments. CCS, YLK, CYL, PKH, FC,
RL helped in sample collection and clinical data retrieval. CSC, KYW, RLHY, DYJ helped
in data analyses. All authors were involved in the writing and final approval of the
manuscript.

Acknowledgements

This work was supported by The University of Hong Kong Seed Funding Programme for
Basic Research (Project Code: 201011159064), and the Hong Kong Research Grants Council
General Research Fund (Ref. 763409M) awarded to C.S.C. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of
the manuscript.